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An imbalance between oxidants and antioxidants is considered a major factor in the development of pulmonary vascular diseases. Oxidative stress seen in pulmonary vascular cells is regulated by increased expression of prooxidant enzymes (e.g., nicotinamide adenine dinucleotide phosphate reduced oxidases) and/or decreased production of antioxidants and antioxidant enzymes (e.g., superoxide dismutases). We and others have shown that expression of antioxidant genes in pulmonary artery cells is regulated by epigenetic mechanisms. In this study, we investigate the regulation of oxidative stress in pulmonary artery cells using inhibitors of histone deacetylases (HDACs). Human pulmonary artery endothelial cells (HPAECs) and human pulmonary artery smooth muscle cells were exposed to an array of HDAC inhibitors followed by analysis of anti- and prooxidant gene expression using quantitative RT-PCR and quantitative RT-PCR array. We found that exposure of HPAECs to scriptaid, N-[4-[(hydroxyamino)carbonyl]phenyl]-α-(1-methylethyl)-benzeneacetamide, and trichostatin A for 24 hours induced expression of extracellular superoxide dismutase (EC-SOD) up to 10-fold, whereas expression of the prooxidant gene NADPH oxidase 4 was decreased by more than 95%. We also found that this differential regulation of anti- and prooxidant gene expression resulted in significant attenuation in the cellular levels of reactive oxygen species. Induction of EC-SOD expression was attenuated by the Janus kinase 2 protein kinase inhibitor AG490 and by silencing Janus kinase 2 expression. Augmentation of EC-SOD expression using scriptaid was associated with increased histone H3 (Lys27) acetylation and H3 (Lys4) trimethylation at the gene promoter. We have determined that oxidative stress in pulmonary endothelial cells is regulated by epigenetic mechanisms and can be modulated using HDAC inhibitors.

The human c10orf10 gene product, also known as decidual protein induced by progesterone (DEPP), is known to be differentially regulated in mouse tissues in response to hypoxia and oxidative stress, however its biological function remains unknown. We found that mice lacking extracellular superoxide dismutase (EC-SOD) show attenuated expression of DEPP in response to acute hypoxia. DEPP mRNA levels, as well as the activity of a reporter gene expressed under the control of the DEPP 5'-flanking region, were significantly upregulated in Hep3B and Vero cells overexpressing EC-SOD. Subcellular fractionation and immunofluorescent microscopy indicated that overexpressed DEPP is co-localized with both protein aggregates and aggresomes. Further biochemical characterization indicates that DEPP protein is unstable and undergoes rapid degradation. Inhibition of proteasome activities significantly increases DEPP protein levels in soluble and insoluble cytosolic fractions. Attenuation of autophagosomal activity by 3-methyladenine increases DEPP protein levels while activation of autophagy by rapamycin reduced DEPP protein levels. In addition, ectopic overexpression of DEPP leads to autophagy activation, while silencing of DEPP attenuates autophagy. Collectively, these results indicate that DEPP is a major hypoxia-inducible gene involved in the activation of autophagy and whose expression is regulated by oxidative stress., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Extracellular superoxide dismutase (EC-SOD) is the main antioxidant enzyme in the extracellular matrix. We developed transgenic mice to analyze the EC-SOD promoter activity in vivo in real time and to identify the important cis-elements flanking the 5' region of the murine EC-SOD gene. Using this model, we demonstrated that luciferase reporter activity correlates closely with endogenous EC-SOD expression, although several interesting differences were also observed. Specifically, luciferase activity was detected at the highest levels in testes, aorta and perirenal fat. Reporter expression was regulated by interferon gamma, a finding that is in agreement with published endogenous EC-SOD gene expression studies. Thus, the 5'-flanking region of mouse EC-SOD gene is responsible, at least in part, for cell specific and inducible expression., (© 2013.)

Extracellular superoxide dismutase (EC-SOD) is the major antioxidant enzyme present in the vascular wall, and is responsible for both the protection of vessels from oxidative stress and for the modulation of vascular tone. Concentrations of EC-SOD in human pulmonary arteries are very high relative to other tissues, and the expression of EC-SOD appears highly restricted to smooth muscle. The molecular basis for this smooth muscle-specific expression of EC-SOD is not known. Here we assessed the role of epigenetic factors in regulating the cell-specific and IFN-γ-inducible expression of EC-SOD in human pulmonary artery cells. The analysis of CpG site methylation within the promoter and coding regions of the EC-SOD gene demonstrated higher levels of DNA methylation within the distal promoter region in endothelial cells compared with smooth muscle cells. Exposure of both cell types to DNA demethylation agents reactivated the transcription of EC-SOD in endothelial cells alone. However, exposure to the histone deacetylase inhibitor trichostatin A (TSA) significantly induced EC-SOD gene expression in both endothelial cells and smooth muscle cells. Concentrations of EC-SOD mRNA were also induced up to 45-fold by IFN-γ in smooth muscle cells, but not in endothelial cells. The IFN-γ-dependent expression of EC-SOD was regulated by the Janus tyrosine kinase/signal transducers and activators of transcription proteins signaling pathway. Simultaneous exposure to TSA and IFN-γ produced a synergistic effect on the induction of EC-SOD gene expression, but only in endothelial cells. These findings provide strong evidence that EC-SOD cell-specific and IFN-γ-inducible expression in pulmonary artery cells is regulated, to a major degree, by epigenetic mechanisms that include histone acetylation and DNA methylation.

The major pulmonary antioxidant enzyme involved in the protection of the lung interstitium from oxidative stress is extracellular superoxide dismutase (EC-SOD). It has been previously shown that EC-SOD knock-out mice are more susceptible to bleomycin-induced lung injury, however, the molecular mechanism(s) remains unclear. We report here that bleomycin-induced lung damage, in EC-SOD KO mice, is associated with increased hyaluronan release into alveolar fluid. Analysis of hyaluronan synthase gene expression and hyaluronan molecular weight distribution suggested that elevated levels of hyaluronan in the alveolar fluid are mostly due to its release from the interstitium. Our results indicate that EC-SOD attenuates bleomycin-induced pulmonary injury, at least in part, by preventing superoxide-mediated release of hyaluronan into alveolar space., (Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Extracellular superoxide dismutase (EC-SOD) plays an important role in maintaining normal redox homeostasis in the lung. It is expressed at very high levels in pulmonary fibroblasts, alveolar type II epithelial cells, and smooth muscle cells. The molecular mechanisms governing this cell-specific expression of EC-SOD are mostly unknown. In our previous studies we showed that EC-SOD cell-specific expression was not attributable to differential transcriptional regulation, suggesting that other, possibly epigenetic, mechanisms are involved in regulation of its expression. In this paper, we show high levels of promoter methylation in A549 cells and correspondingly low levels of methylation in MRC5 cells. Inhibition of DNA methyltransferase activity by 5-azacytidine in A549 cells reactivated EC-SOD transcription (2.75+/-0.16-fold, P<0.001), demonstrating the importance of methylation in the repression of EC-SOD expression. Furthermore, methylation of cytosines in the promoter markedly decreased Sp1/Sp3-driven promoter activity to 30.09+/-2.85% (P<0.001) compared to unmethylated promoter. This attenuation of transcription of the promoter/reporter construct was, at least in part, attributable to the binding of the methyl-binding protein MeCP2 in the insect cells. However, no binding of MeCP2 or MBD2 protein to the EC-SOD promoter was detected in mammalian cells in vivo. We also found marked differences in the chromatin organization of the EC-SOD promoter between these two cell lines, further supporting the important role epigenetic modifications play in the regulation of EC-SOD expression., (2010 Elsevier Inc. All rights reserved.)

Extracellular superoxide dismutase (SOD3), a secretory copper-containing antioxidant enzyme, plays an important role in various oxidative stress-dependent cardiovascular diseases. Although cofactor copper is required for SOD3 activity, it remains unknown whether it can regulate SOD3 transcription. We previously demonstrated that SOD3 activity requires the copper chaperone antioxidant-1 (Atox1), involved in copper delivery to SOD3 at the trans-Golgi network (TGN). Here we show that copper treatment in mouse fibroblasts significantly increases mRNA and protein levels of SOD3, but not SOD1, which is abolished in Atox1-deficient cells. Copper promotes Atox1 translocation to the nucleus. Promoter deletion analysis identifies copper- and Atox1-response elements (REs) at the SOD3 promoter. Gel-shift and ChIP assays reveal that Atox1 directly binds to the Atox1 RE in a copper-dependent manner in vitro and in vivo. Adenovirus-mediated reexpression in Atox1(-/-) cells of nucleus-targeted Atox1 (Atox1-NLS), but not TGN-targeted Atox1 (Atox1-TGN), increases SOD3 transcription without affecting SOD3 activity. Importantly, reexpression of both Atox1-NLS and Atox1-TGN together, but not either alone, in Atox1(-/-) cells increases SOD3 activity. SOD3 transcription is positively regulated by copper through the transcription factor function of Atox1, whereas the full activity of SOD3 requires both the copper chaperone and the transcription factor functions of Atox1. Thus, Atox1 is a potential therapeutic target for oxidant stress-dependent cardiovascular disease.

The molecular mechanisms that govern the transcription of human extracellular superoxide dismutase (EC-SOD), the major extracellular antioxidant enzyme, are largely unknown. To elucidate the mechanisms involved in human EC-SOD gene regulation and expression, we localized multiple transcription start sites to a finite region located 3.9 kb upstream of the ATG initiation codon. Within this segment, we subcloned a 2.7-kb fragment upstream of a luciferase reporter gene; the resulting construct exhibited strong in vivo promoter activity in two lung-derived cell lines. Deletion analysis of the EC-SOD 5'-flanking sequences identified a minimal 0.3-kb region that had strong basal promoter activity. Computer sequence analysis revealed a putative Sp1-like binding site within the EC-SOD proximal promoter region that lacked a TATA-box and showed a high frequency of GC nucleotides. Binding of Sp1 and Sp3 transcription factors to the EC-SOD promoter was confirmed by DNase I footprint analysis, electophoretic mobility shift assay, and competition and supershift assays. Cotransfection of the EC-SOD promoter-luciferase reporter constructs with plasmids encoding Sp1 and Sp3 into Sp-deficient insect SL2 cells showed strong activation of luciferase gene expression. The occupancy of the EC-SOD promoter by Sp1/Sp3 and RNA polymerase II in vivo was determined by chromatin immunoprecipitation assay and correlated well with levels of EC-SOD expression in lung epithelial cells (A549) and pulmonary fibroblasts (MRC5). Collectively, our results demonstrate the important role Sp1 and Sp3 plays in regulating the expression of human EC-SOD in the lung.

This study investigates the role of extracellular SOD (EC-SOD), the major extracellular antioxidant enzyme, in skeletal muscle ischemia and reperfusion (I/R) injury. Pedicled cremaster muscle flaps from homozygous EC-SOD knockout (EC-SOD-/-) and wild-type (WT) mice were subjected to 4.5-h ischemia and 90-min reperfusion followed by functional and molecular analyses. Our results revealed that EC-SOD-/- mice showed significantly profound I/R injury compared with WT littermates. In particular, there was a delayed and incomplete recovery of arterial spasm and blood flow during reperfusion, and more severe acute inflammatory reaction and muscle damage were noted in EC-SOD-/- mice. After 90-min reperfusion, intracellular SOD [copper- and zinc-containing SOD (CuZn-SOD) and manganese-containing (Mn-SOD)] mRNA levels decreased similarly in both groups. EC-SOD mRNA levels increased in WT mice, whereas EC-SOD mRNA was undetectable, as expected, in EC-SOD-/- mice. In both groups of animals, CuZn-SOD protein levels decreased and Mn-SOD protein levels remained unchanged. EC-SOD protein levels decreased in WT mice. Histological analysis showed diffuse edema and inflammation around muscle fibers, which was more pronounced in EC-SOD-/- mice. In conclusion, our data suggest that EC-SOD plays an important role in the protection from skeletal muscle I/R injury caused by excessive generation of reactive oxygen species.

Background: Acute RT-induced damage to the lung is characterized by inflammatory changes, which proceed to the development of fibrotic lesions in the late phase of injury. Ultimately, complete structural ablation will ensue, if the source of inflammatory/fibrogenic mediators and oxidative stress is not removed or attenuated. Therefore, the purpose of this study is to determine whether overexpression of extracellular superoxide dismutase (EC-SOD) in mice ameliorates acute radiation induced injury by inhibiting activation of TGFbeta1 and downregulating the Smad 3 arm of its signal transduction pathway., Methods: Whole thorax radiation (single dose, 15 Gy) was delivered to EC-SOD overexpressing transgenic (XRT-TG) and wild-type (XRT-WT) animals. Mice were sacrificed at 1 day, 1 week, 3, 6, 10 and 14 weeks. Breathing rates, right lung weights, total/differential leukocyte count, activated TGFbeta1 and components of its signal transduction pathway (Smad 3 and p-Smad 2/3) were assessed to determine lung injury., Results: Irradiated wild-type (XRT-WT) animals exhibited time dependent increase in breathing rates and right lung weights, whereas these parameters were significantly less increased (p < 0.05) at 3, 6, 10 and 14 weeks in irradiated transgenic (XRT-TG) mice. An inflammatory response characterized predominantly by macrophage infiltration was pronounced in XRT-WT mice. This acute inflammation was significantly attenuated (p < 0.05) in XRT-TG animals at 1, 3, 6 and 14 weeks. Expression of activated TGFbeta1 and components of its signal transduction pathway were significantly reduced (p < 0.05) at later time-points in XRT-TG vs. XRT-WT., Conclusion: This study shows that overexpression of EC-SOD confers protection against RT-induced acute lung injury. EC-SOD appears to work, in part, via an attenuation of the macrophage response and also decreases TGFbeta1 activation with a subsequent downregulation of the profibrotic TGFbeta pathway.

Hypoxia and biological responses to hypoxia are commonly encountered in both normal and pathologic cellular processes. Here we report that extracellular superoxide dismutase (EC-SOD) plays a major role in regulating the magnitude of hypoxia-induced erythropoietin (Epo) gene expression, thus implicating superoxide as an intermediary signal transduction molecule critical to this process. We found that mice which have the EC-SOD gene inactivated show a marked more than 100-fold elevation in hypoxia-induced Epo gene expression, compared with wild-type controls, which was both dose and time dependent. These mice also showed a significant increase in serum Epo levels after 1 d hypoxia. Interestingly, despite elevated Epo levels, reciprocal changes in hematocrit and reticulocyte counts were not found, suggesting that this newly synthesized Epo lacks functional hematopoietic effects. When EC-SOD was overexpressed in Hep3B cells, we found a significant reduction in Epo gene induction by both CoCl2 (50 microM) and hypoxia (1% O2). Similar findings were noted with another hypoxia-inducible gene, carbonic anhydrase IX. We conclude that EC-SOD functions as a major repressor of hypoxia-induced Epo gene expression, which implicates superoxide as a signaling intermediate whose downstream effects, at least in part, may be mediated by HIF-1alpha.

Extracellular superoxide dismutase (EC-SOD) is the major extracellular antioxidant enzyme and may play a critical role in the pathogenesis of a variety of pulmonary, neurological, and cardiovascular diseases. We report here that exposure to the deacetylase inhibitor trichostatin A (TSA) induces EC-SOD mRNA levels in mIMCD3 and Hepa 1-6 cells, but reduces EC-SOD mRNA levels in MLg cells. To determine the molecular mechanism of TSA-mediated EC-SOD gene regulation, we analyzed EC-SOD's proximal promoter region, which revealed two previously unknown but putative Sp1 cis elements. Transfection of systematically truncated 5'-flanking sequences revealed that the second Sp1 binding site contributes up to 70% of the constitutive EC-SOD promoter activity. Binding of Sp1 and Sp3 transcription factors to this region was confirmed by DNase I footprinting, electrophoretic mobility shift assay, super-shift assay, and chromatin immunoprecipitation. A dominant-negative Sp1 construct considerably reduced EC-SOD promoter activity in mammalian cells, whereas coexpression of Sp1 and Sp3 greatly enhanced reporter activity in SL2 cells. An EC-SOD promoter-reporter construct showed from 5- to 14-fold induction after exposure to TSA, whereas deletion of the Sp1 binding site significantly reduced reporter activation. These results are consistent with Sp1/Sp3 transcription factors providing essential TSA-dependent and basal transcription of the EC-SOD gene and may represent a novel pharmacological pathway for regulating EC-SOD levels in tissue.

Purpose: The purpose of this study was to determine if radiation-induced lung injury is associated with prolonged oxidative stress, and whether chronic overexpression of extracellular superoxide dismutase (EC-SOD) in the lung of transgenic mice protects against radiation-induced lung injury., Methods and Materials: Whole-lung radiation was delivered to EC-SOD overexpressing B6C3 transgenic (XRT-TG) mice and wild-type littermates (XRT-WT). Pulmonary function was assessed by breathing frequency. Right lung wet weight was used as a gross indicator of lung damage. Histopathology was used to assess collagen deposition and tissue fibrosis according to an established grading system. Immunohistochemistry was used to stain and quantify the number of macrophages. ELISA was used to measure activated TGF-beta1. Oxidative stress was assessed by measuring lipid oxidation products (malondialic acid) by HPLC., Results: Four of six XRT-WT mice required euthanasia at 15-19 weeks postradiation because of respiratory distress, whereas no XRT-TG mouse developed distress. All assessments of lung damage at 15-20 weeks postradiation were higher for XRT-WT mice compared with the XRT-TG mice, including breathing frequency (380 vs. 286 bpm, p

We tested the hypothesis that targeted transgenic overexpression of human extracellular superoxide dismutase (EC-SOD) would preserve alveolar development in hyperoxia-exposed newborn mice. We exposed newborn transgenic and wild-type mice to 95% oxygen (O2) or air x 7 days and measured bronchoalveolar lavage cell counts, and lung homogenate EC-SOD, oxidized and reduced glutathione, and myeloperoxidase. We found that total EC-SOD activity in transgenic newborn mice was approximately 2.5x the wild-type activity. Hyperoxia-exposed transgenic mice had less pulmonary neutrophil influx and oxidized glutathione than wild-type littermates at 7 days. We measured alveolar surface and volume density in animals exposed to 14 days more of air or 60% O2. Hyperoxia-exposed transgenic EC-SOD mice had significant preservation of alveolar surface and volume density compared with wild-type littermates. After 7 days 95% O2 + 14 days 60% O2, lung inflammation measured as myeloperoxidase activity was reduced to control levels in all treatment groups.

Extracellular superoxide dismutase (EC-SOD or SOD3) is an important protective enzyme against the toxicity of superoxide radicals that are produced under both physiological and pathophysiological conditions. We have isolated and characterized over 11 kb of the mouse EC-SOD gene and its 5'- and 3'-flanking regions. The gene consists of two exons, with the entire coding region located within exon 2. In order to study the mechanism of cell-specific gene regulation for mouse EC-SOD, we characterized 2500 bp of its 5'-flanking region using cultured cells derived from mouse lung fibroblasts (MLg), kidney medulla (mIMCD3) and hepatocytes (Hepa 1-6). Real-time PCR showed that basal expression of EC-SOD was considerably higher in MLg cells compared with the other cell types. Reporter-gene assays revealed that the proximal promoter region was sufficient to support this high expression in MLg cells. Although no obvious TATA box was identified, our results show that a highly purine-rich region from -208 to +104 contains active binding sites for both the Kruppel-like and Ets families of transcription factors. Using electrophoretic mobility shift, DNase footprinting and reporter gene assays, we identified myeloid zinc finger 1 and gut-enriched Kruppel-like-factor-like nuclear transcription factors as repressors of EC-SOD expression, whereas nuclear transcription factors from the Ets family, such as Elf-1 and GA-binding protein alpha and beta, were potent activators of EC-SOD transcription. We propose a model that highlights competition between Ets activators and Kruppel-like repressors within the proximal promoter region that determines the level of EC-SOD expression in a particular cell type.

Excessive production and/or inadequate removal of reactive oxygen species, especially superoxide anion (O(2)(*-)), have been implicated in the pathogenesis of many cardiovascular diseases, including atherosclerosis, hypertension, diabetes, and in endothelial dysfunction by decreasing nitric oxide (NO) bioactivity. Since the vascular levels of O(2)(*-) are regulated by the superoxide dismutase (SOD) enzymes, a role of SOD in the cardiovascular disease is of substantial interest. Particularly, a major form of SOD in the vessel wall is the extracellular SOD (ecSOD). This review will discuss the characteristics of ecSOD and the role of ecSOD in cardiovascular diseases.

Superoxide dismutases are an ubiquitous family of enzymes that function to efficiently catalyze the dismutation of superoxide anions. Three unique and highly compartmentalized mammalian superoxide dismutases have been biochemically and molecularly characterized to date. SOD1, or CuZn-SOD (EC 1.15.1.1), was the first enzyme to be characterized and is a copper and zinc-containing homodimer that is found almost exclusively in intracellular cytoplasmic spaces. SOD2, or Mn-SOD (EC 1.15.1.1), exists as a tetramer and is initially synthesized containing a leader peptide, which targets this manganese-containing enzyme exclusively to the mitochondrial spaces. SOD3, or EC-SOD (EC 1.15.1.1), is the most recently characterized SOD, exists as a copper and zinc-containing tetramer, and is synthesized containing a signal peptide that directs this enzyme exclusively to extracellular spaces. What role(s) these SODs play in both normal and disease states is only slowly beginning to be understood. A molecular understanding of each of these genes has proven useful toward the deciphering of their biological roles. For example, a variety of single amino acid mutations in SOD1 have been linked to familial amyotrophic lateral sclerosis. Knocking out the SOD2 gene in mice results in a lethal cardiomyopathy. A single amino acid mutation in human SOD3 is associated with 10 to 30-fold increases in serum SOD3 levels. As more information is obtained, further insights will be gained.

The mechanism of tissue injury after exposure to air pollution particles is not known. The biological effect has been postulated to be mediated via an oxidative stress catalyzed by metals present in particulate matter (PM). We utilized a transgenic (Tg) mouse model that overexpresses extracellular superoxide dismutase (EC-SOD) to test the hypothesis that lung injury after exposure to PM results from an oxidative stress in the lower respiratory tract. Wild-type (Wt) and Tg mice were intratracheally instilled with either saline or 50 microg of residual oil fly ash (ROFA). Twenty-four hours later, specimens were obtained and included bronchoalveolar lavage (BAL) and lung for both homogenization and light histopathology. After ROFA exposure, EC-SOD Tg mice showed a significant reduction in BAL total cell counts (composed primarily of neutrophils) and BAL total protein compared with Wt. EC-SOD animals also demonstrated diminished concentrations of inflammatory mediators in BAL. There was no statistically significant difference in BAL lipid peroxidation; however, EC-SOD mice had lower concentrations of oxidized glutathione in the BAL. We conclude that enhanced EC-SOD expression decreased both lung inflammation and damage after exposure to ROFA. This supports a participation of oxidative stress in the inflammatory injury after PM exposure rather than reflecting a response to metals alone.

Reactive oxygen and nitrogen species such as superoxide and nitric oxide are released into the extracellular spaces by inflammatory and airway epithelial cells. These molecules may exacerbate lung injury after influenza virus pneumonia. We hypothesized that enhanced expression of extracellular superoxide dismutase (EC SOD) in mouse airways would attenuate the pathological effects of influenza pneumonia. We compared the pathogenic effects of a nonlethal primary infection with mouse-adapted Hong Kong influenza A/68 virus in transgenic (TG) EC SOD mice versus non-TG (wild-type) littermates. Compared with wild-type mice, EC SOD TG mice showed less lung injury and inflammation as measured by significant blunting of interferon-gamma induction, reduced cell count and total protein in bronchoalveolar lavage fluid, reduced levels of lung nitrite/nitrate nitrotyrosine, and markedly reduced lung pathology. These results demonstrate that enhancing EC SOD in the conducting and distal airways of the lung minimizes influenza-induced lung injury by both ameliorating inflammation and attenuating oxidative stress.

Extracellular superoxide dismutase (EC-SOD, or SOD3) is the major extracellular antioxidant enzyme in the lung. To study the biologic role of EC-SOD in hyperoxic-induced pulmonary disease, we created transgenic (Tg) mice that specifically target overexpression of human EC-SOD (hEC-SOD) to alveolar type II and nonciliated bronchial epithelial cells. Mice heterozygous for the hEC-SOD transgene showed threefold higher EC-SOD levels in the lung compared with wild-type (Wt) littermate controls. A significant amount of hEC-SOD was present in the epithelial lining fluid layer. Both Tg and Wt mice were exposed to normobaric hyperoxia (>99% oxygen) for 48, 72, and 84 hours. Mice overexpressing hEC-SOD in the airways attenuated the hyperoxic lung injury response, showed decreased morphologic evidence of lung damage, had reduced numbers of recruited inflammatory cells, and had a reduced lung wet/dry ratio. To evaluate whether reduced numbers of neutrophil infiltration were directly responsible for the tolerance to oxygen toxicity observed in the Tg mice, we made Wt and Tg mice neutropenic using anti-neutrophil antibodies and subsequently exposed them to 72 hours of hyperoxia. Both Wt and Tg neutrophil-depleted (ND) mice have less severe lung injury compared with non-ND animals, thus providing direct evidence that neutrophils recruited to the lung during hyperoxia play a distinct role in the resultant acute lung injury. We conclude that oxidative and inflammatory processes in the extracellular lung compartment contribute to hyperoxic-induced lung damage and that overexpression of hEC-SOD mediates a protective response to hyperoxia, at least in part, by attenuating the neutrophil inflammatory response.

Objective: Myocardial injury after ischemia and reperfusion may be mediated, in part, by oxygen-derived free radicals. In this study the protective effects of extracellular superoxide dismutase overexpression were directly assessed in the hearts of transgenic mice, after ischemia and reperfusion injury, using an isolated work-performing murine heart preparation and computerized analysis of functional data., Methods: A blinded study was performed to compare cardiac function in the hearts of both transgenic mice with a 3.5-fold overexpression of myocardial extracellular superoxide dismutase (n = 6, 22 to 26 gm) and littermate controls (n = 8, 22 to 26 gm). Preload-dependent cardiac output, contractility, heart rate, stroke work, and stroke volume were evaluated in the two groups before and after a 6-minute period of normothermic ischemia., Results: No differences were found between extracellular superoxide dismutase hearts and control hearts in any parameter of myocardial function before ischemia. After ischemia, decreases in cardiac output occurred in both groups; however, this decrease was larger in control mice compared with extracellular superoxide dismutase mice. A higher percentage of recovery was also observed in the contractility, heart rate, stroke work, and stroke volume of extracellular superoxide dismutase hearts compared with control hearts., Conclusion: After global normothermic ischemia and subsequent reperfusion, decreases in cardiac function occurred in both extracellular superoxide dismutase and control mice; however, a higher percentage of recovery was observed in the extracellular superoxide dismutase overexpressed hearts. These data suggest that extracellular superoxide dismutase transgene overexpression significantly improves preservation of myocardial function after ischemia and reperfusion injury.

Extracellular superoxide dismutase (EC-SOD) is the major extracellular antioxidant enzyme. We have determined the primary structure of mouse EC-SOD by characterization of complementary DNA (cDNA) clones and by amino-acid sequence analysis of purified protein. cDNA sequence analysis indicates that mouse EC-SOD is synthesized as a 251-amino-acid precursor protein with a predicted molecular weight of 27,400 D. Amino-terminal micro sequence analysis of purified mature mouse lung EC-SOD demonstrated the sequence to begin with SSFDLADRLDPV-. These results indicate that EC-SOD as initially synthesized contains a 24-amino-acid precursor peptide, and that the mature protein is 227 amino acids in length. Computer algorithms that predict the most likely site of cotranslational signal peptidase cleavage suggest that processing will occur between amino acids 18 and 19 or 20 and 21, which implies that EC-SOD may be initially synthesized as a pre-pro-protein. Like human EC-SOD, mature mouse EC-SOD is glycosylated. The full-length mouse EC-SOD cDNA is 1,834 base pairs long and is 82% (79% for protein) identical to rat EC-SOD, but only 60% (60% for protein) identical to human EC-SOD. The mouse EC-SOD gene locus (Sod3) was mapped by interspecific backcross haplotype analysis as being 0.9 +/- 0.9 centimorgans distal to the Qdpr locus on mouse Chromosome 5, a position suggesting that the human homologue of EC-SOD will map close to the human QDPR locus (4p15.3). Of nine tissues examined by Northern blot analysis, those of the kidney and lung are by far the major tissues that express EC-SOD messenger RNA. Using in situ hybridization in the mouse lung, we demonstrate EC-SOD gene expression to be highly localized to alveolar Type II epithelial cells. These data suggest that alveolar Type II cells play a central role in mediating EC-SOD antioxidant function in the lung.

Using a reverse transcriptase polymerase chain reaction (RT-PCR), a complement DNA encoding secreted superoxide dismutase (s-SOD) of a mouse kidney has been isolated and the nucleotide sequence was determined. The deduced amino acid sequence of mouse s-SOD cDNA shares 79% identity with the rat seminal SOD sequence and 61% identity with the human SOD3 sequence. Northern blot analysis showed that mouse s-SOD is intensely expressed in the kidney and lung tissues and detectable in other tested tissues, including the brain. The mouse s-SOD gene was assigned to chromosome 5 using fluorescence in situ hybridization analysis and PCR analysis of mouse/hamster hybrid cells.

Background: Myocardial injury after ischemia and reperfusion may be mediated, at least in part, by oxygen-derived free radicals; this supposition is supported by the observation that significant quantities of these radicals are generated during reperfusion. To directly assess the protective effect of extracellular superoxide dismutase (EC-SOD), this study was designed to investigate the ability of EC-SOD overexpression in the hearts of transgenic mice to protect myocardial tissue against ischemiareperfusion injury by use of an isolated work performing murine heart preparation and functional analysis., Methods and Results: Ten transgenic mice (EC.SOD, 28 to 31 g) were studied and compared with 10 control mice (Ctl, 28 to 31 g) in terms of preischemic and postischemic myocardial function. All hearts underwent cardiac harvest and arrest, followed by instrumentation and subsequent reperfusion with warm Krebs-Henseleit solution. Preload-dependent functional analysis was then performed to evaluate cardiac output, contractility (dP/dt), heart rate, stroke work, and stroke volume before and after a 7-minute period of warm ischemia. Results are expressed as mean +/- SEM (ANOVA, paired unpaired t tests). There was no significant difference in preischemic myocardial performance for Ctl and EC-SOD mice. After warm ischemia, cardiac output in EC-SOD was significantly improved compared with Ctl (EC-SOD, 4.55 +/- 0.37 mL/min; Ctl, 2.55 +/- 0.28 mL/min; P < .05). Postischemic dP/dt, stroke work, and stroke volume were also significantly improved in EC-SOD compared with Ctl mice (EC-SOD, 1808 +/- 39 mm Hg/s, 745 +/- 67 dyne.cm, and 13.1 +/- 1.2 microL, respectively; Ctl, 1497 +/- 87 mm Hg/s, 472 +/- 83 dyne.cm, and 8.2 +/- 1.5 microL; P < .05)., Conclusions: EC-SOD overexpressed mice showed significant improvement in postischemic cardiac function compared with Ctl mice. Thus, EC-SOD overexpressed hearts are less susceptible to mild degrees of ischemia-reperfusion injury than normal hearts.

We have isolated and characterized over 10,000 bp of the human EC SOD gene (SOD3 or EC 1.15.1.1) and its 5'- and 3'-flanking regions. Human genomic Southern blot analysis supports the existence of a single gene, without evidence for pseudogenes. The human EC SOD gene spans approximately 5900 bp. The gene can be divided into 3 exons and 2 introns. The 720-bp coding region is uninterrupted and located within exon 3. The 560 bp 5' to the transcription start site were sequenced. No obvious TATA box was identified. A variety of conserved cis elements were identified by database searching. Exon 3 is surrounded by an Alu-J repetitive element in reverse orientation at the 5' and by an Alu-Sx repetitive element in the 3'-flanking DNA. The relative levels of EC SOD tissue-specific expression were determined by RNA gel blot analysis. Adult heart, placenta, pancreas, and lung had the most expression, followed by kidney, skeletal muscle, and liver. Little EC SOD message was found in the brain. A second unique mRNA, approximately 4.2 kb in length, was highly expressed in skeletal muscle. When tissue enzyme activity is compared to relative mRNA levels, there is a marked disparity in the brain, pancreas, and lung, suggesting that these tissues have enhanced affinity for circulating EC SOD or translate the EC SOD message more efficiently than other tissues. These results indicate that the EC SOD gene contains unique transcriptional regulatory elements and that its expression may be regulated at the post-transcriptional or post-translational level. The characterization of the human EC SOD gene should now allow the development of further insights into its biology and provide the basis for studies of its role in human heritable disorders.